Zika Virus (ZIVR) is a very rapidly emerging infectious disease that has been associated with microcephaly in the children of infected mothers in an ongoing outbreak in Brazil. ZIVR is similar to other flavaviruses, such as Dengue, Yellow Fever, and West Nile, which are transmitted to humans by mosquitos. Controlling ZIVR from a public health perspective and appropriately managing individual patients will require that the diagnosis of ZIVR infection can be made rapidly and accurately. Because the symptoms of ZIVR infection are similar to those of other infections, the diagnosis cannot be made on clinical grounds alone and a diagnostic assay is required. Unfortunately, no routine and accurate assay for the diagnosis of ZIVR infection is currently available. Existing assays are either limited to specialized laboratories or give false positive results in patients that have been infected with other flaviviruses. We propose to significantly advance the development of a ZIVR diagnostic assay by generating antibodies that specifically recognize ZIVR, but not other flavaviruses, and using these to construct a prototype ZIVR antigen ELISA assay. Virus-specific antibodies are the key reagents required for the most widely used types of diagnostic assays, which detect the presence of viral proteins in the blood. Such assays have the advantage of yielding rapid results and being applicable to almost any health care setting. However, to be accurate, such assays must use antibodies that are specific for a single virus. Here, we will use single-chain antibody phage display technology to generate antibodies specific to the NS1 protein produced by ZIVR. We have chosen NS1 because it has proven to be an excellent diagnostic target for other viral infections such as Dengue. The specific procedures that will be used include immunizing mice against ZIVR NS1, harvesting the antibody-producing B cells from these mice, and using them to make a library of single-chain antibodies that are expressed on the tips of a bacteriophage (viruses that infect bacteria). This phage library will then be screened for antibody clones that bind to ZIVR, but not to related viruses using standard phage display procedures. Once antibody phage clones with the appropriate binding characteristics are identified, they will be converted into full-length antibodies suitable for use in a diagnostic assay. At all stages of the process, candidate antibodies will be tested to validate their sensitivity and specificity for ZIVR NS1. The successful completion of this project will result in the availability of validated high affinity highly specific anti-ZIVR NS1 Abs that can be used to construct sensitive and specific ZIVR diagnostic assays. We will also develop a ZIVR NS1 ELISA suitable for clinical testing in the course of these studies. This will markedly improve our ability to accurately diagnose ZIVR infection, thus significantly improving our capacity to track, control, and appropriately treat this disease.